Panel A shows western blots of KPC cells treated with cET-ASONT or cET-ASOKras, indicating phosphorylation levels of EPHA2Ser897, p90RSK, and ERK1/2. Panel B displays the effect of RSK inhibitor LHJ685 on EPHA2 Ser897 phosphorylation. Panel C is a line graph showing Kras messenger RNA expression levels in KPC cells treated with varying concentrations of cET-ASOKras. Panel D presents western blots of Epha2 knockout KPC cells treated with ccET-ASONT or cET-ASOKras. Panel E is a heatmap of messenger RNA expression levels of the scavenger receptor family in Epha2 wild-type and Epha2 knockout KPC cells. Panel F shows fluorescence micrographs of cET-ASOKras internalization and EPHA2 and CD44 localization in KPC cells. Panel G is a line graph showing fluorescence intensity across a distance. Panel H is a scatter plot of fluorescence polarization of cET-ASO-Cy3 or hyaluronic acid-Cy3 in the presence of recombinant human CD44. Panel I is a bar chart showing displacement of cET-ASO-Cy3 from the cET-ASO-Cy3/recombinant human CD44 complex by unlabeled hyaluronic acid. Panel J shows fluorescence micrographs of proximity ligation between cET-ASO and CD44 in KPC cells. Panel K is a line graph quantifying the average number of proximity ligation assay spots per cell. Panel L shows a pull-down assay of biotin-labeled CD44 from EPHA2-TurboID-expressing cells. Panel M presents fluorescence micrographs of internalized cET-ASOKras and biotin-labeled targets in EPHA2-TurboID-expressing KPC cells. Panel N shows western blots of cET-ASOKras-induced activation of ERK1/2, p90RSK, and EPHA2 in Cd44 knockout KPC cells. Panel O shows fluorescence micrographs of intracellular CD44 and internalized ASO in control and Cd44 knockout KPC cells. Panel P is a bar chart showing Kras messenger RNA expression in non-targeting or Cd44 CRISPR knockout KPC cell lines treated with cET-ASOKras.
SRs trigger EPHA2-dependent uptake and trafficking of ASOs. (A) Nontargeting cET-ASO (cET-ASONT) or cET-ASOKras induction of EPHA2 Ser897, p90RSK, and ERK1/2 phosphorylation in KPC cells. GAPDH is a loading control. (B) Effect of the RSK inhibitor LHJ685 (RSKi) on the cET-ASOKras -induced phosphorylation of EPHA2 Ser897 in KPC cells. (C)Kras mRNA expression 72-h following treatment with the indicated concentrations of cET-ASOKras (72 h) in vehicle (gray line) or RSK inhibitor-treated (10 μM, magenta line) KPC cells; data are mean ± SEM, n = 3 independent experiments. (D) cET-ASONT or cET-ASOKras induction of p90RSK and ERK1/2 phosphorylation in Epha2−/− KPC cells. Western blotting was performed on the same samples that were used for the experiment presented in supplemental Fig. S2 C. Thus, the vinculin blot used as a loading control in Fig. 3 D is also used as a sample control for supplemental Fig. S2 C. (E) Heatmap showing the levels of mRNA expression of the SR family in either Epha2+/+ (top) or Epha2−/− (bottom) KPC cells. (F and G) Internalization of cET-ASOKras (16 h, red), EPHA2 (green), and CD44 (white) in KPC cells. (H) Fluorescence polarization of either cET-ASO-Cy3 or HA-Cy3 (10 kDa) in the presence of the indicated concentrations of recombinant-hCD44. Receptor–ligand affinity data are fitted using a 1:1 binding model. Fitting (R2) and dissociation values (KD) are shown for each molecule; data are mean ± SEM, n = 3 independent experiments. (I) Displacement of cET-ASO-Cy3 from the cET-ASO-Cy3/rhCD44 complex by increasing concentrations of unlabelled HA (10 kDa) is shown; data are mean ± SEM, n = 3 independent experiments. (J and K) Fluorescence micrographs of Duolink proximity ligation (PLA, red) between cET-ASO and CD44 in KPC cells (K), and quantification of the average number of PLA spots per cell after cET-ASO addition for 4 or 16 h (K). Data are expressed as log10(x+1) spots per cell, n = 3 independent experiments, ANOVA Tukey post hoc test. (L) Pulldown of biotin-labelled CD44 from EPHA2-TurboID expressing cells after cET-ASOKras treatment for 16 h. (M) cET-ASOKras-positive internalized vesicles (red) and biotin-labelled targets (green) in EPHA2-TiD expressing KPC cells. (N) cET-ASOKras–induced activation of ERK1/2, p90RSK and EPHA2 phospho-Ser897 in Cd44-knockout KPC cells. GAPDH is a loading control. (O) Overlap of intracellular CD44 and internalized ASO in control and Cd44KO KPC cells. (P)Kras mRNA expression in either nontargeting or Cd44CRISPR-KO KPC cell lines treated with the indicated concentrations of cET-ASOKras (72 h), n = 5 independent experiments, TWA Sidak for all panels. Source data are available for this figure: SourceData F3. TWA, two-way ANOVA.